Asthma and rhinitis are atopic (allergic) diseases affecting between 20-30% of the population. They are associated with acute and chronic inflammatory responses resulting from contact with protein particles in the environment. Initially, exposure of the airway immune system to otherwise innocuous aeroallergens elicits specific immune responses leading ultimately to production of IgE in predisposed individuals (Togias (2003) J. Allergy Clin. Immunol. 111:1171-1183; Braunstahl and Hellings (2003) Curr. Opin. Pulm. Med. 9:45-51). Subsequent cross-linking of IgE by these allergens is implicated in acute allergic rhinitis and asthma exacerbations. In those predisposed individuals, allergen exposure results in activation of antigen-specific CD4+ T lymphocytes of the Th2 phenotype; secretion of specific cytokines, including IL-4, IL-5, and IL-13; production of IgE; priming of mast cells; and the recruitment of eosinophils (Wills-Karp (1999) Annu. Rev. Immunol. 17:255-281). Th2 cells regulate immune responses by releasing these cytokine mediators into the local environment and via direct cell-cell interactions (Agnello, et al. (2003) J. Clin. Immunol. 23:147-161; Leigh, et al. (2004) Am. J. Respir. Crit. Care Med. 169:860-867; Bochner and Busse (2004) J. Allergy Clin. Immunol. 113:868-875).
Experimental animal models of allergen-induced asthma, in which the profile of cytokines present in the airways can be manipulated, support a role for Th2 cytokines in asthma pathogenesis (see, e.g., Cohn, et al. (1998) J. Immunol. 161:3813-3816; Hogan, et al. (1998) J. Immunol. 161:1501-1509; Kuperman, et al. (1998) J. Exp. Med. 187:939-948; Wills-Karp, et al. (1998) Science 282:2258-2261). In murine models of experimental asthma, Th2 cytokines promote airway inflammation and eosinophilia, mucus production, and airway hyperresponsiveness. Taking into account differences in genetic backgrounds and redundancy in cytokine function there is general agreement regarding the role of IL-4, IL-5 and IL-13 in asthma pathogenesis (Wills-Karp (1999) supra; Foster, et al. (2002) Pharmacol. Ther. 94:253-264). IL-4 is primarily responsible for the development of CD4+ T cells with a Th2 phenotype (Kopf, et al. (1993) Nature 362:245-248; Le Gros, et al. (1990) J. Exp. Med. 172:921-929; McKenzie, et al. (1998) Immunity 9:423-432). IL-5 is required for eosinophil maturation and activation (Campbell, et al. (1987) Proc. Natl. Acad. Sci. USA 84:6629-6633; Clutterbuck, et al. (1987) Eur. J. Immunol. 17:1743-1750). IL-13 alone is capable of inducing airway hyperresponsiveness in naïve mice (Wills-Karp, et al. (1998) supra; Grunig, et al. (1998) Science 282:2261-2263), even in the complete absence of airway eosinophils (Mattes, et al. (2002) J. Exp. Med. 195:1433-1444). Moreover, IL-13 is also implicated in airway remodeling. Stable pulmonary expression of IL-13 induces epithelial cell hypertrophy, mucus cell metaplasia and subepithelial collagen deposition (Zhu, et al. (1999) J. Clin. Invest. 103:779-788).
Both IL-4 and IL-13 activate receptors that share the IL-4 receptor alpha (IL-4R alpha) subunit, which induces activation of STAT-6, an SH2 domain containing transcription factor that regulates gene expression (Hou, et al. (1994) Science 265:1701-1706; Quelle, et al. (1995) Mol. Cell. Biol. 15:3336-3343). Binding of IL-4 or IL-13 to their receptors induces activation of the cytokine receptor associated tyrosine kinases Jak 1, Jak 3 and Tyk 2 (Hershey (2003) J. Allergy Clin. Immunol. 111:677-690). These kinases in turn phosphorylate specific tyrosine residues on the IL-4R alpha subunit. Cytoplasmic STAT-6 is recruited to the phosphorylated receptor via the STAT-6 SH2 domain whereupon it is in turn phosphorylated by the receptor associated Jak/Tyk tyrosine kinases. Phosphorylated STAT-6 molecules then dissociate from the receptor, form homodimers via interactions between STAT-6 SH2 domains and phosphotyrosine residues on paired molecules. Only after tyrosine phosphorylation and homodimerization can STAT-6 translocate to the nucleus and regulate IL-4/IL-13-dependent gene expression. Following sensitization and challenge with allergen, STAT-6 knockout mice do not develop the characteristic airway hyperresponsiveness and lung pathology associated with asthma (Kuperman, et al. (1998) supra; Akimoto, et al. (1998) J. Exp. Med. 187:1537-1542). Recent data from murine models of experimental asthma suggest that the inability of STAT-6 knockout mice to develop asthma pathogenesis may be due to the loss of IL-13 activity (Wills-Karp, et al. (1998) supra; Grunig, et al. (1998) supra; Mattes, et al. (2001) J. Immunol. 167:1683-1692; Walter, et al. (2001) J. Immunol. 167:4668-4675; Pope, et al. (2001) J. Allergy Clin. Immunol. 108:594-601; Kuperman, et al. (2002) Nat. Med. 8:885-889; U.S. Pat. No. 5,866,760) although IL-4-mediated effects may also play a role.
Th2 cytokines, their receptors, and the transcription factors that mediate Th2 cytokine-specific cellular responses are therapeutic targets for the treatment of allergic rhinitis and asthma. One therapeutic approach that has shown promise is to inhibit expression of the proteins that regulate asthma pathogenesis. In experimental asthma, inhibiting expression of IL-4, or the common beta chain shared by IL-5, IL-3 and GM-CSF receptors, or the Th2-specific GATA-3 transcription factor using antisense oligonucleotides effectively inhibits airway inflammatory responses as well as airway hyperresponsiveness in experimental asthma (Allakhverdi, et al. (2002) Am. J. Respir. Crit. Care Med. 165:1015-1021; Finotto, et al. (2001) J. Exp. Med. 193:1247-1260; Molet, et al. (1999) J. Allergy Clin. Immunol. 104:205-214). Further, the inhibition of STAT-6 expression using antisense oligonucleotides is taught in WO 98/40478.
In addition to antisense technology, soluble cytokine receptor subunits have been used to bind to and sequester IL-4 or IL-13 to inhibit allergic asthma (Wills-Karp, et al. (1998) supra; Grunig, et al. (1998) surpa; Henderson, et al. (2000) J. Immunol. 164:1086-1095; Borish, et al. (2001) J. Allergy Clin. Immunol. 107:963-970). In addition, dominant negative mutants of IL-4 and IL-13 effectively inhibit activation of IL-4/IL-13 receptors by the wild-type cytokines and thus may also have therapeutic potential (Oshima and Puri (2001) FASEB J. 15:1469-1471; Hahn, et al. (2003) J. Allergy Clin. Immunol. 111:1361-1369). Likewise, deletion mutants of STAT-6 have been generated which are attenuated or function as dominant negative variants which decrease STAT-6 dimerization (U.S. Pat. No. 6,368,828).
A peptide composed of the protein transduction domain from antennapedia coupled to the sequence surrounding tyrosine residue 606 (Tyr-606) of the human IL-4R alpha subunit has also been produced (Stolzenberger, et al. (2001) Eur. J. Biochem. 268:4809-4814). This peptide inhibits IL-4-induced tyrosine phosphorylation of STAT-6, although the effect is only transient. Moreover, in vivo activity of this peptide was not disclosed.
Methods for identifying agents which modulate the interaction between STAT-6 and its receptor are taught in U.S. Pat. No. 6,207,391. Agents are identified in competitive binding assays with high affinity receptor peptides of the sequence Tyr-Xaa1-Xaa2-Xaa3 (SEQ ID NO:1), wherein Xaa1 is Lys, Val, Arg, Ile, or Met; Xaa2 is Pro, Ala, or Ser; Xaa3 is Trp, Tyr, Phe, His, or Leu; and the N-terminal tyrosine is phosphorylated.
Further, WO 01/83517 teaches dipeptide derivatives which bind STAT-6 for use as immunomodulators, U.S. Pat. No. 6,426,331 teaches small molecules which modulate the function of STAT proteins, and WO 02/038107 discloses additional compounds for modulating STAT-6 signaling.